Nanoporous membrane immunosensor

ABSTRACT

A sensor for a selected analyte in a test sample has (a) a semipermeable membrane with pores for retaining the analyte, where the membrane has been chemically modified by attachment of membrane modifiers; (b) immunoassay labels which have label binding ligands where these label binding ligands will have a binding affinity for the membrane modifiers in the presence of the analyte, and a measurably different binding affinity for the membrane modifiers in the absence of the analyte; and (c) a label detecting system, for detecting the presence of the labels on the membrane.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to assays and more specificallyto binding assays, such as antibody/hapten or DNA interactions, usingnanoporous membranes for increasing the local concentration of theanalyte, to improve the overall sensitivity of the assay. This techniquecan be used with a wide range of labeling schemes, including radiolabeling and labeling with magnetic beads.

2. Description of the Related Art

Binding assays, for example immunoassays, are widely used in the food,medical, and pharmaceutical industries as diagnostic tests for a widerange of target molecules. Many binding assays have been produced andmarketed since the principle was first developed.

Immunoassays typically exploit the binding capabilities of antibodies.Antibodies are protein molecules which are frequently consideredfighters of infections. They fight infections by binding to theinfectious material in a specific manner, forming a complex. This is asignal to the organism to reject that complex. Antibodies may also beproduced to specifically bind to a wide range of compounds, as a keyfits a lock. However other molecules (e.g., chelators, strands ofpolynucleic acids, receptors including cellular receptors) that arecapable of recognizing and selectively binding other molecules may beemployed to detect a wide range of species, such as polynucleic acids(DNA or RNA), polypeptides, glycolipids, hormones, polymers, metal ions,and certain low molecular weight organic species including a number ofillegal drugs. To be useful in an assay, this recognition event mustgenerate a signal that is macroscopically observable. The methodemployed to generate such a signal is one way of distinguishing thevarious types of immunoassays.

The first immunoassay used radioactive labeling. This radioimmunoassay(RIA) is quite sensitive and widely used, but the hazards, expense, andrestrictions associated with handling radioactive material makesalternative immunoassays desirable. Recently, enzyme and fluorescenceassays have replaced radioimmunoassays. The present inventors and othershave developed techniques using magnetic beads as labels forimmunoassays. Other known immunoassay labeling techniques use colloidsor fluorescent dyes.

An ongoing goal of immunoassay development is improving the lower limitof detection (LLD). Likewise, it is an ongoing goal of immunoassaydevelopment to shorten processing time. This is particularly true inorder to counter threats of biological warfare and terrorism, as well asother field applications.

Solid supports are used in many immunoassays, typically as adsorbentlayers. Many of these, such as nylon and nitrocellulose membranes havepore sizes greater than 25 nm, to amplify the signal by increasing thesurface area of the assay.

Some microbiological assays use membranes to separate and concentratebacteria. These membranes are typically on the order of 200 nm poresize.

Viruses have been identified with aluminum ultrafiltration membraneswith 20 nm pores. Organisms immobilized on these membranes have beenidentified using both specific and nonspecific dyes.

Chemically selective membranes are used in some chemical sensors to passthe analyte through the membrane. Larger molecules are not allowed topass through the membrane into the internal sensing solution.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to selectively detect awide range of target species, with a high degree of sensitivity.

It is a further object of this invention to selectively detect a widerange of target species, with a short processing time.

It is a further goal of this invention to improve sensitivity to 100 to1000 times that of the current laboratory standard enzyme-linkedimmunosorbant assay (ELISA), with processing times at least 10 timesshorter than ELISA.

These and additional objects of the invention are accomplished by thestructures and processes hereinafter described.

An aspect of the present invention is a sensor for a selected analyte ina test sample having (a) a semipermeable membrane with pores forretaining the analyte, where the membrane has been chemically modifiedby attachment of membrane modifiers; (b) immunoassay labels which havelabel binding ligands where these label binding ligands will have abinding affinity for the membrane modifiers in the presence of theanalyte, and a measurably different binding affinity for the membranemodifiers in the absence of the analyte; and (c) a label detectingsystem, for detecting the presence of the labels on the membrane.

Another aspect of the invention is a method for detecting an analyte ina test sample, having the steps: (a) modifying a side of a semipermeablemembrane, the membrane having pores for retaining the analyte, withmembrane modifiers; (b) placing the test sample in contact with themembrane on the side of the membrane with the membrane modifiers; (c)drawing the test sample through the membrane, osmotically or with theapplication of differential pressure across the membrane, so that anyanalyte present in the test sample is drawn towards the modifiedmembrane surface; (d) disposing immunoassay labels on the side of themembrane with the membrane modifiers, where these labels have labelbinding ligands where these label binding ligands will have a bindingaffinity for the membrane modifiers in the presence of the analyte, anda measurably different binding affinity for the membrane modifiers inthe absence of the analyte; and (e) detecting the presence of theimmunoassay labels on the membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention will be obtained readilyby reference to the following Description of the Preferred Embodimentsand the accompanying drawings in which like numerals in differentfigures represent the same structures or elements, wherein:

FIG. 1 is a schematic representation of a membrane for a preferredembodiment of the invention.

FIG. 2 is a schematic representation of a preferred embodiment of theinvention.

FIG. 3 is a schematic representation of a fluid handling system for apreferred embodiment of the invention.

FIG. 4 is a schematic representation of a preferred embodiment of theinvention using the force differentiation assay as the labeling anddetection technique.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following are incorporated by reference herein, in their entireties,for all purposes: (a) U.S. Pat. No. 5,807,758, and (b) Ser. No.09/008,782.

The term test sample, as used herein, refers to a material suspected ofcontaining the analyte. The test sample can be used directly as obtainedfrom the source or following a pre-treatment to modify the character ofthe sample. The test sample can be derived from any biological source,such as physiological fluid including, but not intended to be limited toblood, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine,milk, ascites fluid, mucous, synovial fluid, peritonaeal fluid, amnioticfluid and the like; fermentation broths; cell cultures; chemicalreaction mixtures and the like. The test samples can be pretreated priorto use, such as preparing plasma from blood, diluting viscous fluids,and the like. Methods of treatment can involve filtration, distillation,concentration, inactivation of interfering components, and the additionof reagents. In addition to biological or physiological fluids, otherliquid samples can be used such as water, food products, aerosolcollectors and the like for the performance of environmental or foodproduction assays. In addition, a solid material suspected of containingthe analyte can be used as the test sample. In some instances, it may bebeneficial to modify a solid test sample to form a liquid medium or torelease the analyte.

Binding members, such as the analytes and the membrane and labelmodifiers of the invention, refers to a member of a binding pair, i.e.,two different molecules wherein one of the molecules specifically bindsto the second molecule through chemical or physical means. In additionto the well-known antigen and antibody binding pair members, otherbinding pairs include, but are not intended to be limited to, biotin andavidin, carbohydrates and lectins, complementary nucleotide sequences,complementary peptide sequences, effector and receptor molecules,enzymes cofactors and enzymes, enzyme inhibitors and enzymes, a peptidesequence and an antibody specific for the sequence or the entireprotein, polymeric acids and bases, dyes and protein binders, peptidesand specific protein binders (e.g., ribonuclease, S-peptide andribonuclease S-protein), sugar and boronic acid, and similar moleculeshaving an affinity which permits their associations in a binding assay.Furthermore, binding pairs can include members that are analogs of theoriginal binding member, e.g., an analyte-analog or binding member madeby recombinant techniques or molecular engineering. If the bindingmember is an immunoreactant it can be, e.g., an antibody, antigen,hapten, or complex thereof, and if an antibody is used, it can be amonoclonal or polyclonal antibody, a recombinant protein or antibody, achimeric antibody, a mixture(s) or fragment(s) thereof, as well as amixture of an antibody and other binding members. The details of thepreparations of such antibodies, peptides and nucleotides and theirsuitability for use as binding members in a binding assay are well-knownto those skilled-in-the-art.

The term analyte or analyte of interest, as used herein, refers to thecompound or composition to be detected or measured and which has atleast one epitope or binding site. The analyte can be any substance forwhich there exists a naturally occurring binding member or for which abinding member can be prepared. Analytes include, but are not limitedto, toxins, organic compounds, proteins, peptides, microorganisms, aminoacids, carbohydrates, nucleic acids, hormones, steroids, vitamins, drugs(including those administered for therapeutic purposes as well as thoseadministered for illicit purposes), virus particles and metabolites ofor antibodies to any of the above substances. For example, such analytesinclude, but are not intended to be limited to ferritin; creatininekinase MIB (CK-MIB); digoxin; phenyloin; phenobarbitol; carbamazepine;vancomycin; gentamycin; theophylline; valproic acid; quinidine;leutinizing hormone (LH); follicle stimulating hormone (FSH); estradiol,progesterone; IgE antibodies; vitamin B2 micro-globulin; glycatedhemoglobin (Gly. Hb); cortisol; digitoxin; N-acetylprocainamide (NAPA);procainamide; antibodies to rubella, such as rubella-IgG andrubella-IgM; antibodies to toxoplasmosis, such as toxoplasmosisIgG(Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM); testosterone;salicylates; acetaminophen; hepatitis B virus surface antigen (HBsAg);antibodies to hepatitis B core antigen, such as anti-hepatitis B coreantigen IgG and IgM (Anti-BBC); human immune deficiency virus 1 and 2(HTLV); hepatitis B e antigen (HBeAg); antibodies to hepatitis B eantigen (Anti-Hbe); thyroid stimulating hormone (TSH); thyroxine (T4);total triiodothyronin (Total T3); free triiodiothyronin (Free T3);carcinoembryoic antigen (CEA); and alpha fetal protein (AF); and drugsof abuse and controlled substances, including but not intended to belimited to, amphetamine; methamphetamine; barbituates such asamobarbital, seobarbital, pentobarbital, phenobarbital, and barbital;benzodiazepines such as librium and valium; cannabinoids such as hashishand marijuana; cocaine; fetanyl; LSD; methapualone; opiaets such asheroin, morphine, codine, hydromorphone, hydrocodone, methadone,oxycodone, oxymorphone and opium; phencyclidine; and propoxyhene. Theterm analyte also includes any antigenic substances, haptens,antibodies, macromolecules and combinations thereof.

Preferably, the pore diameter of the membranes of the present inventionis selected to retain the particular analyte of interest. Preferably,the pore diameter of the membranes of the present invention is selectedto pass potential interferents that are smaller than the analyte ofinterest. For some embodiments, it will be preferred that the porediameter does not exceed 25 nm. For other embodiments, it will bepreferred that the pore diameter does not exceed 10 nm. This issufficient to retain most analytes of interest. For larger analytes ofinterest, e.g. bacteria, it will be preferred to have pore diameters onthe order of about 100 nm, to retain bacteria but to pass viruses. Foreven larger analytes of interest, e.g. pollen, spores, and dustparticles, it will be preferred to have pore diameters on the order ofabout 1000 nm, to retain these particles but to pass bacteria andviruses.

Referring to FIG. 1, in a preferred embodiment of the invention, asemipermeable membrane 10 has membrane modifiers 12 (typicallyantibodies) bound to the membrane through linkers 14. These linkers canbe chosen from a wide range of linkers used in surface chemistry, buttypically will be hydrophillic polymer films. The pores 16 preferablyhave nominal diameters less than 25 nm.

The membrane modifiers may be the same across the surface of themembrane, or several different types of membrane modifiers may bepatterned in an array, to allow for parallel processing within a singletest vessel.

Referring to FIG. 2, this membrane may be used to rapidly transport theanalyte 18 to the binders if a differential or osmotic pressure isapplied across the membrane. This pressure, from a pressure source (notshown) such as a pump, causes bulk flow from a volume on one side of themembrane 48 to a volume on the other side of the membrane 50, asindicated by the dashed arrows. If a differential pressure source isused (as distinct from osmotic pressure), the pressure may be positiveor negative. That is, the pump may be configured to increase thepressure on the side of the membrane with the test sample, or todecrease the pressure on the distal side of the membrane. A labeledbinder 20 is then used to sense the binding event using a detector (notshown in this figure).

The labeled binders 20 may be the same, or several different types oflabeled binders may be patterned used, typically in conjunction with amembrane patterned with several different membrane modifiers in anarray, to allow for parallel processing within a single test vessel.

Details of alternative assay configurations are described below.

Ultrafiltration Membranes for Sensing

The rate of a chemical reaction at a surface such as a membrane iscontrolled by at least three phenomena. First, the reactant must betransported to a surface, which is a process that can be controlled bydiffusion or convection. If pressure is applied across the membrane,mass transport will be dominated by the convective flow of the sampletowards the membrane. The second phenomenon, controlling the overallreaction rate, is the kinetics of the reaction at the interface. In thecase of an antibody-antigen interaction, this reaction can be writtenB+A⇄B−Awhere B is the binder, A is the analyte and B-A is the binder-analytecomplex. The amount of bound analyte is determined by the equilibriumconstant of the reactionK _(eq) =k ₁ /k ⁻¹ =[B−A]/[B][A]where k₁ and k⁻¹ are the forward and reverse rate constants,respectively. The third phenomenon controlling the rate of reaction isthe diffusion of the analyte away from the surface. The advantage ofusing membranes is that if the membrane retains a significant portion ofan analyte, the analyte will be rapidly concentrated at the membranesurface resulting in an increase in the amount of analyte bound.Conventional membrane-based immunoassays bind the analyte in theirmicron size pores producing an active area 1-100 microns deep in themembrane. The ultrafiltration membrane in this invention has a pore sizeof 25 nm or less, which acts to concentrate the analyte at the surfaceof the membrane. The observed increase in sensitivity of the assayexecuted on the ultrafiltration membranes in this invention isattributed to the higher concentration of analyte in the active area ofthe detector.

Ultrafiltration membranes are widely used for separation andpurification processes. The pore size of the membrane, the filtrationpressure and the physical properties of the species determine theefficiency of retention of a species. Many of these membranes arecomposed of organic polymeric materials. Nucleopore membranes, anexample of a polymer ultrafiltration membrane, are composed ofpolycarbonate, have pore sizes of 10-10,000 nm and pore densities ofapproximately 10¹² m⁻² (3). Several inorganic ultrafiltration membranesare also available: (i) Anopore (membranes which are anodically etchedfrom aluminum, have pore sizes ranging from 10-250 nm, and pore densityof 10¹²-10¹⁵ m⁻² (4); (ii) Nanochannel glass membranes are drawn fromoptical fibers and have similar properties to the Anopore membrane;(iii) Microfabricated membranes currently have larger pore sizes and lowdensity of pores than the Anopore membranes.

For this invention, it is desirable to have an ultrafiltration membranethat has a high density of pores to avoid the need for high differentialpressures. In addition, for applications with optical detection it isimportant that the membrane be flat, optically translucent and notlikely to change shape or size during the course of the assay. Organicultrafiltration membranes do not meet these requirements. The inorganicmembranes have the physical properties required for this assay: (i) highflow rates with moderate pressures; (ii) optically translucent membranesthat retain their shape when wet; (iii) high retention efficiencies formacromolecules. Studies of 10 nm pore size Anopore membranes atpressures of 100 kPa or less indicate that 6, 20 and 66 percent of30,000, 67,000 and 150,000 Da proteins are retained, respectively.

Methods of Preparing Activated Membranes

The ultrafiltration membrane in this invention must be functionalizedwith a binder in order to act as a sensor. However, fouling is aphenomenon that will severely limit the use of ultrafiltration membranesfor sensing. Protein fouling has been attributed to adsorption, poreplugging and cake consolidation. We have minimized the effect of proteinfouling by executing the immunoassay on a dense hydrophilic polymer filmthat inhibits protein adsorption. In a preferred embodiment, the activesurface of an Anopore membrane was coated with a dense layer ofbiotinylated poly (ethylene glycol) (PEG) using a polyethylene imine(PEI) adhesion layer (see Example 1). Many variations on this chemistrycan be used: (i) The surface can be activated using several differentapproaches, e.g., silanization or thiolation. (ii) Other hydrophilicpolymers could be used to inhibit protein adsorption, e.g., dextran.(iii) Other approaches to inhibit nonspecific protein adsorption areavailable, e.g., adsorption of proteins such as bovine serum albumin(BSA). The biotin-PEG functionalized membranes have the followingadvantages: (i) the membranes can be stored in a dry form for extendedperiods of time; (ii) the concentration of receptors on the membrane canbe varied; (iii) the surface can be regenerated. It is especiallydesirably to make these membranes reusable due to their cost.

Many types of binders can be immobilized on the membrane. Bindersagainst specific analytes may be either directly or indirectly bound toa hydrophilic polymer film. Preferred binders include DNAoligonucleotides, PNA oligonucleotides, polyclonal antibodies andmonoclonal antibodies. In a preferred embodiment, antibodies arespecifically immobilized on the biotinylated PEG surface usingantibody-streptavidin conjugates (see Example 3). Alternatively, bindersmay be directly bound to the hydrophilic polymeric films functionalizedwith N-hydroxysuccinimide (NHS), maleimide, or vinyl groups. Forexample, antibodies can be thiolated with N-succinimidylS-acetylthioacetate and then reacted with α-vinyl sulfone,ω-n-hydroxysuccinimide PEG, 3,000 MW functionalized polymers. There areof course many variations on this chemistry.

Assay Methods

The assay method will be composed of at least three steps: (i)preparation of the sample, (ii) reaction of the analyte at the membranesurface and (iii) detection of the binding events.

Those skilled in the art will recognize that complex samples may requirepre-purification and/or cellular disruption of bacteria. For example, wehave found that immunoassays involving bacteria have superiorsensitivity if the bacteria are disrupted using either chemical ormechanical means. In the preferred embodiment all samples would beexposed to ultrasonic power in the presence of inorganic particles (SeeExample 4 below).

Those skilled in the art will realize that there are numerousconfigurations in which the analyte can be bound and detected. Threeexamples are described in schemes 1-3. Scheme 1: Direct Sandwich Capturemembrane Complex Membrane-binder analyte-binder-label Membrane-biotinstreptavidin-binder-analyte-binder-label

This assay can be executed in a series of sequential steps on themembrane or the complex could be formed in solution and then bound onthe membrane. Note that convective transport may be used at each step ofthe assay to enhance the response time. Scheme 2: Indirect SandwichCapture membrane Complex Membrane-binderanalyte-binder-antibinder-binder-label Membrane-biotinstreptavidin-binder-analyte-antibinder-binder-label

In this case, it would probably be most convenient to execute the assayon the membrane. Again, convective transport may be used at each step ofthe assay to enhance the response time.

Scheme 3: Competitive

In some instances the analyte might have only one epitope available forbinding. In this case a competitive assay may be used to detect theanalyte. Labeled analyte would be added to the sample and itsdifferential binding would be detected.

In these assay configurations the label is used to produce a signal thatis related to the amount of analyte in the sample. The signal producingsystem can be composed of one or more members. The measurement of thissignal will normally involve electromagnetic radiation absorption oremission. The signal producing systems can be any suitable system,including chromogens, catalyzed reactions, chemiluminesence, andradioactive labels. In the preferred embodiment the forcedifferentiation assay will be used to detect the analyte, see Example 2.

Apparatus

The assay apparatus will be composed of a membrane, fluid handlingsystem, and detector. A fluid handling system and detector are describedin this section.

The differential pressure necessary to transport the analyte to themembrane surface may be produced osmotically, or with either positivepressure or negative pressure from a pump. In cases in which the samplewill be handled batch wise conventional vacuum filtration equipment or asyringe filtration apparatus may be used (see examples).

Biosensors typically require automated processing and continuousoperation. A schematic of a cell for performing continuous assays isshown in FIG. 3. This 1×0.25×0.5″ 21 cell is drawn with five reactionchambers 22, 24, 26, 28, 30 in which the sample passes from left toright. This cell makes it possible to run five sequential assays if eachsurface used one time. If the surfaces can be regenerated, numerousassays can be run in a single cell. In this cell the active area of eachchamber is patterned with four binders 32, 34, 36, 38. Patterning asurface with binders has the advantage that multiple analytes can bedetected while simultaneously running internal standards. Techniques forpatterning binders on solid surface are well known to those skilled inthe art. A preferred embodiment would be to use ink jet technology toaccurately distribute small volumes of binder-streptavidin conjugates onspecific areas of biotin-PEG functionalized membrane 10. This cellincludes a manifold 40 that will allow the sample to be channeled to theactive area of the membrane and pass through the cell. Clearly, this isnot the only geometry in which this cell could be constructed.

Labeling Techniques

As noted above, the labeling system can be any suitable system forlabeling in immunoassays. Suitable systems include those using labelssuch as chromogens, catalyzed reactions, chemi-luminesence, radioactivelabels, and magnetic beads. In a preferred embodiment the forcedifferentiation assay (Ser. No. 09/008,782) will be used to detect theanalyte.

Chromogens

Chromogens include compounds which absorb light in a distinctive range,so that a color may be observed, or emit light when irradiated withlight of a particular wavelength or wavelength range e.g. fluorescers.

Colloidal particles such as nanometer scale gold are excellentchromogens. Further, magnetic-polymer composites will also adsorb light.

The choice of dye may be varied widely, being primarily chosen toprovide an intense color with minimum absorption by the immunosorbingzone support. Illustrative dye types include quinoline dyes,triarylmethane dyes, acridine dyes, alizarin dyes; phthaleins, insectdyes, azo dyes, anthraquinoid dyes, cyanine dyes, phenazathionium dyes,and phenazoxonium dyes.

A wide variety of fluorescers may be employed either by themselves or inconjunction with quencher molecules.

Fluorescers of interest fall into a variety of categories having certainprimary functionalities. These primary functionalities include 1- and2-aminoaphthalene, p,p′-diaminostilbenes, pyrenes,quaternaryphenanthridine salts, 9-aminoacridines,p,p′-diaminobenzophenone imines, anthracenes, oxacarbocyanine,merocyanine, 3-aminoequilenin, perylene, bis-benzoxazole, bis-p-oxazolylbenzene, 1,2-benzophenazin, retinol, bis-3-aminopyridinium salts,hellebrigenin, tetracycline, sterophenol, benzimidazolylphenylamine,2-oxo-3-chromen, indole, xanthene, 7-hydroxycoumarin, phenoxazine,salicylate, strophanthidin, porphyrins, triarylmethanes and flavin.

Individual fluorescent compounds which have functionalities for linkingor can be modified to incorporate such functionalities include dansylchloride, fluoresceins such as 3,6-dihydroxy-9-phenylxanthhydrol,rhodamineisothiocyanate, N-phenyl 1-amino-8-sulfonatonaphthalene,N-phenyl 2-amino-6-sulfonatonaphthalene,4-acetamido-4-isothiocyanatostilbene-2,2′-disulfonic acid,pyrene-3-sulfonic acid, 2-toluidinonaphthalene-6-sulfonate, N-phenyl,N-methyl 2-aminonaphthalene-6-sulfonate, ethidium bromide, atebrine,auromine-0,2-(9′-anthroyl)palmitate, dansyl phosphatidylethanolarmine,N,N′-dioctadecyl oxacarbocyanine, N,N′-dihexyl oxacarbocyanine,merocyanine, 4-(3′-pyrenyl)butyrate, d-3-aminodesoxyequilenin,12-(9′-anthroyl)stearate, 2-methylanthracene, 9-vinylanthracene,2,2′-(vinylene-p-phenylene)-bis-benzoxazole,p-bis[2-(4-methyl-5-phenyloxazolyl)]benzene,6-dimethylamino-1,2-benzophenazin, retinol, bis(3′-aminopyridinium)1,10-decandiyl diiodide, sulfonaphthylhydrazone of hellebrigenin,chlortetra-cycline, N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide, N-[p-(2-benzimidazolyl)-phenyl]maleimide, N-(4-fluoranthyl)maleimide, bis(homovanillic acid), resazarin,4-chloro-7-nitro-2.1.3-benzooxadiazole, merocyanine 540, resorufin, rosebengal, and 2,4-diphenyl-3(2H)-furanone.

Fluorescers are preferred to absorptive dyes to the extent that a singlefluorescer can provide for multiplication of a signal. By irradiating afluorescer with light, one can obtain a plurality of emissions. Thus, asingle label can provide for a plurality of measurable events.

Various other combinations and protocols could be employed dependingupon the nature of the analyte.

Catalysis

Both enzymatic and nonenzymatic catalysts may be employed. Preferably,enzymatic catalysts will be employed, since they frequently provide formore rapid reactions, a desirable versatility in the variety ofreactions, and have well characterized properties.

In choosing an enzyme, there will be many considerations in addition tothose involved with the reaction of interest. These considerationsinclude the stability of the enzyme, the desirability of a high turnoverrate, the sensitivity of the rate to variations in the physicalenvironment, the nature of the substrate(s) and product(s), theavailability of the enzyme, the effect of conjugation of the enzyme onthe enzyme's properties.

Of particular interest in the subject invention is the use of coupledcatalysts, usually two or more enzymes, where the product of one enzymeserves as the substrate of the other enzyme. One or more enzymes arebound to the surface, while one enzyme is always bound to a mip.Alternatively, two enzymes can be bound to a mip and an additionalenzyme may be bound to the surface.

The solute will be the substrate of any one of the enzymes, butpreferably of an enzyme bound to the surface. The enzymatic reaction mayinvolve modifying the solute to a product which is the substrate ofanother enzyme or production of a compound which does not include asubstantial portion of the solute, which serves as an enzyme substrate.The first situation may be illustrated by glucose-6-phosphate beingcatalytically hydrolyzed by alkaline phosphatase to glucose, whereinglucose is a substrate for glucose oxidase. The second situation may beillustrated by glucose formed from glucose-6-phosphate being oxidized byglucose oxidase to provide hydrogen peroxide which would enzymaticallyreact with the signal generator precursor and a peroxide to produce thesignal generator.

Coupled catalysts can also involve an enzyme with a non-enzymaticcatalyst. The enzyme can produce a reactant which undergoes a reactioncatalyzed by the non-enzymatic catalyst or the non-enzymatic catalystmay produce a substrate (includes coenzymes) for the enzyme. Forexample, G6PDH could catalyze the conversion of NAD and G6P to NADHwhich reacts with tetrazolium salts to produce an insoluble dye.

A wide variety of nonenzymatic catalysts which may be employed in thisinvention are found in U.S. Pat. No. 4,160,645, the appropriate portionsof which are incorporated herein by reference. The nonenzymaticcatalysts employ as reactants a first compound which reacts by a1-electron transfer and a second compound which reacts by a 2-electrontransfer, where the two reactants are capable of reacting with eachother slowly, if at all, in the absence of the catalyst.

Various combinations of enzymes may be employed to provide a signalgenerating compound at the surface. Particularly, combinations ofhydrolases may be employed to produce an insoluble signal generator. Asingle hydrolase may act in a substantially equivalent manner to anenzyme pair by employing the appropriate substrate. Alternatively,combinations of hydrolases and oxido-reductases can provide the signalgenerator. Also, combinations of oxidoreductases may be used to producean insoluble signal generator. Usually there will be a preferredcatalyst at the surface, since as indicated previously, by appropriatechoice of the catalyst at the surface, a greater number of reagents maybe combined in a single formulation.

Chemiluminescers

An alternative source of light as a detectible signal is achemiluminescent source. The chemiluminescent source involves a compoundwhich becomes electronically excited by a chemical reaction and may thenemit light which serves as the detectible signal or donates energy to afluorescent acceptor.

A diverse number of families of compounds have been found to providechemiluminescence under a variety of conditions. One family of compoundsis 2,3-dihydro-1,-4-phthalazinedione. The most popular compound isluminol, which is the 5-amino compound. Other members of the familyinclude the 5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benzanalog. These compounds can be made to luminesce with alkaline hydrogenperoxide or calcium hypochlorite and base. Another family of compoundsis the 2,4,5-triphenylimidazoles, with lophine as the common name forthe parent product. Chemiluminescent analogs include para-dimethylaminoand -methoxy substituents. Chemiluminescence may also be obtained withoxalates, usually oxalyl active esters e.g. p-nitrophenyl and a peroxidee.g. hydrogen peroxide, under basic conditions. Alternatively,luciferins may be used in conjunction with luciferase or lucigenins.

Radioactive Levels

Various radioisotope find common use. These include tritium (³H),radioactive iodine (¹²⁵I), radioactive carbon (¹⁴C), radioactivephosphorus (³²P); or the like. Methods for labeling of compounds withradioactive labels are well known in the art.

Magnetic Bead Labeling

Magnetic beads can be used as labels for immunoassays. U.S. Pat. No.5,807,758 describes one method of using magnetic beads as labels, wheremodified beads will selectively bind to a modified cantilever, dependingon whether the analyte is present. An applied magnetic field will exerta force on the cantilever, which may be detected using conventionaltechniques for measuring cantilever deflection.

Preferably, the force differentiation assay, described in co-pendingapplication Ser. No. 09/008,782, is used. In this technique, themembrane and the magnetic beads are modified with specific bindingagents, so that these bead modifiers will have a binding affinity forthe membrane modifiers in the presence of the analyte species, and ameasurably different binding affinity for the membrane modifiers in theabsence of the analyte species. The beads and the test sample areintroduced into the test vessel, and an adjustable magnetic field sourceis used to apply a magnetic force to the beads. An imaging system isused to determine whether the beads are bound to the membrane, therebytesting for the analyte.

Referring to FIG. 4, depicting a sandwich assay configuration for thepresent invention, a nanoporous membrane 10 is disposed in a testvessel, dividing the test vessel into two volumes 48, 50. Magnetic beads19 are disposed in the vessel, within the first volume 48.

The beads 19 are modified with molecules which are referred to herein asbead modifiers 23, and the membrane 10 is modified on the side facingthe first volume with membrane modifiers 12. Both of these types ofmodifiers will be selected from those molecules that are capable ofrecognizing and selectively binding other molecules, includingantibodies, haptens, polynucleic acids, polypeptides, glycolipids,hormones, polymers, metal ions, and certain low molecular weight organicspecies (see above).

The mechanism for applying a variable, normal magnetic field to thebeads 19 is shown here as a movable annular magnet 42. The annularmagnet produces a uniform {right arrow over (B)} field which is orientedalong its axis across millimeter size areas. It has been discovered thata millimeter scale NdFeB magnet can apply a uniform field over sampleareas consistent with imaging by optical microscopy, if the magnet iscarefully centered. This normal B field acts upon the bead 10 to createa normal force (F) on the beads 19, which in turn puts tension on thebond between the bead 19 and the membrane 10. Since covalent bonds aretypically much stronger than specific molecular interactions (such asantibody-hapten interactions), the strength of the linkage between thebead and the membrane is limited by the strength of this specificmolecular interaction.

In addition to annular magnets, other magnetic geometries may be used,such as discs, bars, or other flat shapes, so long as the B field hasthe desired properties in the area under observation. High permeabilityfocusing cones positioned between the magnet and the substrate may beused to shape the field so that the field has a higher gradient. Sincethe force on a paramagnetic bead is related to the field gradient, theuse of these cones will tend to increase the forces acting on the beads.

As the field intensity is varied, eventually a point is reached wherethe bead separates from the substrate, indicating that the force fromthe field has exceeded the strength of nonspecific or specific molecularinteraction. By observing when beads separate from the substrate, onecan observe when this point is reached. It should be noted that thisseparation point will depend not only on the force applied to thelinkage between the bead and the substrate, but also on the observationtime and the system temperature.

A preferred embodiment of the invention further comprises a microscope,typically an optical microscope 44, for imaging beads bound to thesubstrate, and separated from the substrate. Preferably, the microscope44 is connected through connecting electronics (often including a videocamera) 46 to a computer 48 or to a video recorder for analyzing imagesfrom the microscope. The advantage of the annular magnet 42 is that itdoes not interfere with transmitted light in an optical microscope.However, reflected light microscopy makes it possible to use solidmagnet geometries.

For this preferred embodiment of the invention, it will be important tohave the capacity to identify single beads (as distinguished fromaggregates), to count the beads quickly and reliably, and to determinetheir relative position on the substrate. This positional information isadvantageous for several reasons. For example, in some applications itmay be advantageous to pattern the substrate by attaching differenttypes of substrate modifiers on different regions of the substrate. Itwill be advantageous in such applications to identify where on asubstrate a given bead is bound. Accordingly, the imaging system for thepresent invention, including the microscope 44, computer 48, andconnecting electronics 46, should have the capacity to count beads thathave separated from the substrate (or, alternatively, count beads thathave not separated from the substrate). Typically, this will meancapturing a digital image from the microscope (either directly using aframe grabber or indirectly using a video recorder) and analyzing itusing image analysis algorithms on the computer 48.

Additionally, the imaging system for the present invention preferablyhas the capacity to discriminate between single beads on the substrateand clusters of two or more beads on the substrate, based on their size.It has been discovered that non-uniform surface chemistries and magneticfields of the structures taught by Rohr produce the following non-idealbehavior.

Brownian motion causes the beads to move on the surface. This motionleads a significant fraction of the beads to form dimers and aggregates,if the beads are “sticky” (i.e., tend to stay together once they arebrought together). Multibody interactions in these aggregates lead toenhanced magnetization of the clusters when the {right arrow over (B)}field is applied, and greatly accelerates their displacement. Imageanalysis allows one to identify the level of aggregation and correct forits effects. It should be noted that the fraction of beads as monomersand aggregates is strongly related to the amount of analyte on thesurface and the nonspecific adhesive properties of the surfaces;therefore, aggregation can also be used to independently determineanalyte concentration and surface properties.

Furthermore, under all but the most ideal circumstances a fraction(2-20%) of the beads adhere to the surface nonspecifically, even underhigh forces (>2 pN). It has been observed that these beads capture otherbeads that move laterally in the solution, and thus form string shapedaggregates. Image analysis makes it possible to identify theseaggregates and discard them from consideration. Detection techniquesthat measure integrated signals can not distinguish these beads fromspecifically bound beads.

Nonspecific adhesion between beads, and between beads and the membrane,appears to increase when the beads are loaded with proteins, whichsuggests protein-protein interactions are the primary source for thisadhesion.

It has been discovered that a commercially available microscope(Axiovert 100 microscope with a 63× Acroplan objective, Carl Zeiss, OneZeiss Dr., Thornwood, N.Y. 10594), electronics (VE-1000 CCD72black/white video system from DAGE-MTI, Michigan City, Ind.; DT 3152Fidelity PCI frame grabber, Data Translation, 100 Locke Dr, Marlboro,Mass. 01752-1192), image analysis software (Image-Pro Version 2.0, MediaCybernetics, 8484 Georgia Ave., Silver Spring, Md. 20910), and computer(Pentium 66 MHz Computer with 1 GB hard drive) will reliably identifysuperparamagnetic 2.6 micron diameter beads and clusters thereof. Onceclusters have been identified, they can be ignored, i.e. discounted fromfurther analysis. Thus, when the position of the beads is monitored(i.e., monitored for whether the beads are bound or unbound to thesubstrate), only single beads will be analyzed, dramatically improvingthe accuracy of the detector.

It has further been discovered that beads imaged through such amicroscope may be monitored for movement, and that unbound beads willmove over short time scales, a fraction of a second or a few seconds,permitting these beads to be identified as unbound, and likewisediscounted from further analysis.

The magnitude of the adhesive force between the bead and surface isdetermined by several factors, i.e., the magnitude of the nonspecificforces, the number of specific molecular interactions linking the beadto the surface and the manner in which these interactions are stressed.

Surface modification chemistries (described below) have been developedthat consistently produce very low nonspecific adhesive forces in amajority of beads. Typically, 80-98% of the beads can be removed from asurface at force equivalent to their buoyant weight, i.e., ≈40femtoNewtons (fN) in the case of Dynal's M280 beads. The number ofspecific molecular interactions linking a bead to the surface willdepend on the density and flexibility of ligands and receptors on thebead and surface.

EXAMPLES

Having described the invention, the following examples are given toillustrate specific applications of the invention, including the bestmode now known to perform the invention. These specific examples are notintended to limit the scope of the invention described in thisapplication.

Example 1 Preparation Of Functionalized Membrane: Peg Biotin Membranewith Streptavidin-Antibody Conjugate

Anodisc membrane was hydrated with 200 ml of pure water for 1 minute.Excess water was removed, and the membrane was rinsed with 50 mM sodiumbicarbonate buffer (NaHCO₃) pH 8.2. Excess buffer was removed from themembrane and 5% (w/v) PEI in 50 mM sodium bicarbonate buffer (NaHCO₃) pH8.2 was added. Incubation was at room temperature for 1 hour.

The membrane was rinsed three times with water and once with 50 mMsodium bicarbonate buffer (NaHCO₃) pH 8.2. Excess liquid was removedfrom the membrane, followed by the addition of a-biotin, w-NHSpoly(ethylene glycol)-carbonate, MW 3,400 (Shearwater Polymers,Huntsville, Ala.) at 20 mg/mL in 50 mM sodium bicarbonate buffer(NaHCO₃) pH 8.2. The PEG was incubated at room temperature for 2 hours,excess PEG-biotin solution washed off with three rinses of water andstored dry.

A sandwich immunoassay was built on the PEG-biotin functionalizedAnodisc membranes. The membranes were rehydrated with 200 ml of waterfor 1 minute and rinsed with 0.1 M phosphate buffered saline (PBS) pH7.4. Next 1% (w/v) bovine serum albumin (BSA) (Sigma Chemical Company,St. Louis, Mo.) was added to the surface for 1 hour to blocknon-specific sites. The membrane was then washed 3 times using PBS with0.05% Tween 20 (PBST). Next, the appropriate antibody-streptavidinconjugates were diluted in PBST and added to the membrane in 200 mlvolumes. The optimal conjugate concentration was determined by serialdilution analysis. Rabbit antibody-streptavidin for B. globigii and Goatantibody-streptavidin for ovalbumin and MS-2 were used as the captureantibodies. The conjugates were incubated on the surface for 1 hour,then excess antibodies were removed by washing 3 times using PBST andrinsing once with PBS. The surfaces can be stored in PBS at 4 C for upto 12 hrs.

Example 2 Ovalbumin (OVA) Assay

A functionalized membrane was incubated with the appropriate dilution ofgoat antibody—(ovalbumin)-streptavidin conjugate (200 ml) and thenwashed with PBST to remove unbound antibodies as described in Example 1.

The membrane was then placed into a glass microanalysis vacuum membraneholder (Fisher Scientific, Springfield, N.J.) composed of a borosilicateglass funnel, base, fritted glass support, spring clamp, and No. 5stopper. A 1 mL sample of a standard solution of known concentration(analytes=Ova ng/ml=0, 10, 1, 0.1, 0.01) was added to the filtrationholder and incubated for 5-10 minutes. The analyte solution was thenfiltered using a water aspirator for 5 minutes. Unbound analyte wasremoved by washing the membrane 3 times with PBST. A sandwichingantibody, rabbit-antibody-ovalbumin (affinity purified), was then addedto the membrane (200 μl) at 2 μg/ml in PBST and incubated for 1 hour.The more ovalbumin present in the test solution the more 2^(nd) antibodywill bind. The membrane was then washed three times with PBST, andrinsed with PBS. The membrane was then placed onto a glass microscopeslide, excess fluid was removed, and anti-rabbit IgG-Seramag beadsdiluted in 0.1% (w/v) BSA were added to the membrane and incubated for30 minutes.

The samples were analyzed using a Carl Zeiss Axiovert 100 TV invertedmicroscope fitted with a motorized stage (Ludl Electronics, N.Y.). Acustom written software program operated the reader, this enabledcomputer control of hardware via a serial port. The image analysis wasperformed in real time by accessing routines provided by Image Pro plus,version 3.0 commercial imaging software (Media Cybernetics). Themembrane was analyzed in 3 positions consisting of a 128×96 mm area at adistance of 200 μm apart using a water immersion objective (63×), 0.9numerical aperture (NA) (Carl Zeiss) before and after exposure to themagnet. A magnetic force, was applied using a NdFeB block magnet (MagnetSales and Mfg. Co., Culvert City, Calif.) magnetized perpendicular tothe substrate. The magnet was placed 0.1 mm from the surface for 10seconds to remove unbound beads from the sample surface. The remainingbeads were then counted.

The standard curve was constructed from these data and was expressed asthe percentage of beads bound to the surface after exposure to themagnetic force versus concentration of the analyte. The cut off valuesto determine whether a sample was positive or negative was calculated bytaking the percent bound beads of the negative control, PBST solution,plus three standard deviations. The average binding for the variousanalytes are as follows (ng Ovalbumin): 1 ng=81%, 0.1 ng=41%, 0.01ng=26%, 0.001 ng=1%, 0 ng=2%. The results show a sensitivity of 0.01 ngfor Ovalbumin.

Example 3 MS-2 Assay

A functionalized membrane was incubated with the capture antibody, goatantibody—(MS-2)-streptavidin conjugate, and rinsed with PBST as statedin Example 1. Follow the same procedure as stated in Example 2 withthese few exceptions: capture antibody used was goatantibody-(MS-2)-streptavidin conjugate; analytes=MS-2=0, 10⁵, 10⁴, 10³pfu/ml; sandwiching antibody, Rabbit-antibody—MS-2, was then added tothe membrane (200 μl) at 1 μg/ml in PBST. The average binding for thevarious analytes are as follows (pfu MS2): 0=4%, 10⁵=66.5%, 10⁴=35.4%,10³=22% The results show a sensitivity for MS-2 exceeding 10³ pfu/ml.

Example 4 Bacillus Globigii (BG) Assay

As previously noted, cellular disruption of bacteria, via chemical ormechanical means is preferred before the sample can be added to thereaction chamber. These methods include subjecting the bacterial cellsto a hot detergent treatment, freeze-thaw cycles, beadmill-homogenization, or a commercially available bacterial proteinextraction reagent (B-PER, Pierce). Since B. globigii is a spore formingbacteria, the most effective method of cell disruption is bead millhomogenization (18).

A functionalized membrane was incubated with Rabbitantibody-(BG)-streptavidin conjugate and rinsed with PBST. In order tobreak apart B. globigii, the sample was first subjected to bead millhomogenization (18). Glass beads (0.1-1 mm) were added to a 0.75 ml BGsolution: 1 g glass beads per microcentrifuge tube. The sample was addedto the bead beater (Mini-Bead Beater-8, Bio-Spec products) and mixed on“Homogenize” for 3 minutes. The samples were then centrifuged at12,000×g for 6 minutes. The supernatant was then removed andrecentrifuged for 2 minutes at 12,000×g to eliminate any extraneousglass particles.

A novel method of cellular disruption involving glass beads was alsoused. The analyte solution was added to 1 g of glass beads (0.1-1 mm) ina borosilicate glass culture tube and sonicated using a Branson Sonifier(Branson Ultrasonics Corporation, Danbury Conn.) for 3 minutes. Thesupernatant was removed and added to a microcentrifuge tube andcentrifuged at 12,000×g for 6 minutes.

1 ml of supernatant was removed and added to the filtration apparatusfor incubation with the functionalized surface (analytes=BG=0, 10⁵, 10⁴,10³, 10^(2.5) cfu/ml) for 5-10 minutes and then filtered using a wateraspirator for 5 minutes. Unbound analyte was removed by washing themembrane 3 times with PBST, then a sandwiching antibody,goat-antibody—B. globigii, was added to the membrane (200 μl) at 2 μg/mlin PBST and incubated for 1 hour. The more B. globigii present in thetest solution the more 2^(nd) antibody will bind. The membrane was thenwashed three times with PBST, and rinsed with PBS.

The membrane was then placed on a glass microscope slide, excess fluidwas removed, and anti-goat IgG-Seramag beads diluted in 0.1% (w/v) BSAwere added to the membrane and incubated for 30 minutes. The analysis ofthe samples was the same as in Example 2. The average binding for thevarious analytes are as follows (cfu B. globigii): 0=14%, 10⁵=93%,10⁴=85%, 10³=66%, 10^(2.5)=44%. The sensitivity for B. globigiiexceeding 10^(2.5) cfu/ml

Example 5 Ova in Presence of MS-2

A functionalized membrane was incubated with goatantibody-(ovalbumin)-streptavidin conjugate and rinsed with PBST asdescribed in Example 1. The same procedure as stated in example 2 wasfollowed with these few exceptions: Capture antibody was goatantibody-(ovalbumin)-streptavidin conjugate; analytes=Ova ng/ml=0, 10,1, 0.1, 0.01, Ova 1 ng/ml+MS-2 10⁸ pfu/ml, Ova 1 ng/ml+MS-2 10⁵ pfu/ml,Ova 0.01 ng/ml+MS-2 10⁸ pfu/ml, Ova 0.01 ng/ml+MS-2 10⁸ pfu/ml; thesandwiching antibody, rabbit-antibody-ovalbumin (affinity purified),added was at 2 μg/ml in PBST. The membrane was then washed three timeswith PBST, and rinsed with PBS.

The membrane was then placed on a glass microscope slide, excess fluidwas removed, and anti-rabbit IgG-Seramag beads diluted in 0.1% (w/v) BSAwere added to the membrane and incubated for 30 minutes. The resultsshow a sensitivity of 0.01 ng for Ovalbumin. The total binding decreasesby 32% in the Ova 1 ng/ml+MS-2 10⁸ pfu/ml and the remainder of the mixedanalyte assays show a decrease of 10% in binding. This is most likelycaused by steric hindrance.

Example 6 OVA assay-with Filtration of 2^(nd) Antibody

A functionalized membrane was incubated with goatantibody—(ovalbumin)—streptavidin conjugate and rinsed with PBST asdescribed in example 1. The membrane was then placed into the glassmicrofilter holder and a 1 mL sample (analytes=Ova ng/ml=0, 10, 1, 0.1,0.01) was added to the filtration holder and incubated for 5-10 minutes.The analyte solution was then filtered using a water aspirator for 5minutes. Immediately following filtration of the analyte, thesandwiching antibody, rabbit-antibody-ovalbumin (affinity purified), wasthen added to the membrane at 1 μg (0.5 μg/ml over a 2 ml volume). Thesecondary antibody was passed through the membrane slowly to concentratethe antibody at the interface. The membrane was then washed three timeswith PBST, and rinsed with PBS. The membrane was then placed on a glassmicroscope slide, excess fluid was removed, and anti-rabbit IgG-Seramagbeads diluted in 0.1% (w/v) BSA were added to the membrane and incubatedfor 30 minutes. The membrane was analyzed as described in Example 2. Theresults were as follows (ng Ovalbumin): 0=15%; 1=81%; 0.01=55%. Thesensitivity of the Ovalbumin assay with filtration of 2^(nd) antibodywas 0.01 ng.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

1-14. (canceled)
 15. A sensor for independently detecting a plurality ofanalytes in a test solution, comprising: a test vessel; a semipermeablemembrane with pores for retaining the analyte, dividing said test vesselinto a first volume and a second volume, wherein the surface of saidmembrane is chemically modified by attachment of at least two distinctmembrane surface modifiers on at least a side facing said first volume,wherein said at least two distinct membrane surface modifiers arepatterned into an array of distinct regions on said membrane surface; atleast two distinct groups of immunoassay labels disposed within saidfirst volume, wherein each of said groups of immunoassay labels hasdistinct label binding ligands where these label binding ligands willhave a binding affinity for one of said distinct membrane surfacemodifiers in the presence of the analyte, and a measurably differentbinding affinity for said one of said distinct membrane surfacemodifiers in the absence of the analyte; a pressure source, for drivingsaid test solution from said first volume into said second volume; and alabel detecting system, for detecting the presence or absence of saidlabels in each of said regions on said membrane. 16-25. (canceled)
 26. Asensor for independently detecting a plurality of analytes in a testsolution, comprising: a test vessel; a semipermeable membrane with poresfor retaining the analyte, dividing said test vessel into a first volumeand a second volume, said pores are selected to prevent the analyte frompassing into or through said semipermeable membrane; wherein saidmembrane is chemically modified by attachment of at least two distinctmembrane modifiers on at least a side facing said first volume but notwithin said pores; wherein said at least two distinct membrane modifiersare patterned into an array of distinct regions on said membrane; atleast two distinct groups of assay labels disposed within said firstvolume, wherein each of said groups of assay labels has distinct labelmodifiers, said label modifiers having a binding affinity for one ofsaid distinct membrane modifiers in the presence of the analyte, and ameasurably different binding affinity for said one of said distinctmembrane modifiers in the absence of the analyte; a pressure source, fordriving said test solution from said first volume into said secondvolume; and a label detecting system, for detecting the presence orabsence of said labels in each of said regions on said membrane.
 27. Thesensor of claim 26, wherein said membrane supports a 100 kPa pressureload and said membrane is functionalized with a binder at the surface ofsaid membrane in order for said membrane to act as a sensor.
 28. Thesensor of claim 26, wherein said membrane has pores not greater than 10nm in diameter.
 29. The sensor of claim 26, wherein said membrane has apore density of at least 10¹⁵/m².
 30. The sensor of claim 26, whereinsaid membrane is essentially flat and optically translucent when wet.31. The sensor of claim 30, wherein said membrane remains translucent,and the shape of said membrane remains flat even under pressureassociated with flow of solution through said membrane.
 32. The sensorof claim 26, wherein said membrane is an aluminum oxide membrane. 33.The sensor of claim 26, wherein an active surface of said membrane ismodified with a biotin-polyethylene-glycol (PEG) using apolyethyleneimine (PEI) layer.
 34. The sensor of claim 26, wherein saidpores allow a solvent to pass through while preventing flow of saidbinder, analyte, or assay labels.
 35. The sensor of claim 26, whereinsaid membrane has pores not greater than 20 nm in diameter. 36-45.(canceled)